Bacteria live in capricious environments, in which they must continuously sense external conditions in order to adjust their shape, motility and physiology. The histidine-aspartate phosphorelay signal-transduction system (also known as the two-component system) is important in cellular adaptation to environmental changes in both prokaryotes and lower eukaryotes. In this system, protein histidine kinases function as sensors and signal transducers. The Escherichia coli osmosensor, EnvZ, is a transmembrane protein with histidine kinase activity in its cytoplasmic region. The cytoplasmic region contains two functional domains: domain A (residues 223-289) contains the conserved histidine residue (H243), a site of autophosphorylation as well as transphosphorylation to the conserved D55 residue of response regulator OmpR, whereas domain B (residues 290-450) encloses several highly conserved regions (G1, G2, F and N boxes) and is able to phosphorylate H243. Here we present the solution structure of domain B, the catalytic core of EnvZ. This core has a novel protein kinase structure, distinct from the serine/threonine/tyrosine kinase fold, with unanticipated similarities to both heatshock protein 90 and DNA gyrase B.
We have developed an antisense oligonucleotide microarray for the study of gene expression and regulation in Bacillus subtilis by using Affymetrix technology. Quality control tests of the B. subtilis GeneChip were performed to ascertain the quality of the array. These tests included optimization of the labeling and hybridization conditions, determination of the linear dynamic range of gene expression levels, and assessment of differential gene expression patterns of known vitamin biosynthetic genes. In minimal medium, we detected transcripts for approximately 70% of the known open reading frames (ORFs). In addition, we were able to monitor the transcript level of known biosynthetic genes regulated by riboflavin, biotin, or thiamine. Moreover, novel transcripts were also detected within intergenic regions and on the opposite coding strand of known ORFs. Several of these novel transcripts were subsequently correlated to new coding regions.Gene expression in bacteria has been traditionally analyzed by transcriptional or translational fusions to promoterless "reporter" genes (e.g., lacZ, cat, and gus) or by direct detection of transcripts using Northern blotting or reverse transcription-PCR (RT-PCR). With the completion of many bacterial genomes and the development of large-scale analysis tools such as DNA genomic arrays, however, researchers have increasingly applied genomics tools in their research. Measurements of mRNA levels using genome arrays for Escherichia coli, Bacillus subtilis, Streptococcus pneumoniae, and Haemophilus influenzae (10,11,12,17,23,24,29,32,38,39,41) have been found to offer many advantages to traditional gene-monitoring methods. Since the structure of bacterial genomes is relatively simple, containing ca. 4,000 genes and few repetitive sequences, DNA arrays can monitor transcript levels of an entire genome in a single hybridization with high sensitivity. This can lead to the elucidation of complex interactions among genetic networks, which then can be coupled with results from other newer technologies that analyze global protein synthesis (proteome) and metabolite levels (metabolome) to provide a comprehensive picture of the physiology of the bacterium (13,14,18,34,35,42).Using the public B. subtilis genome sequence (20), we developed an oligonucleotide B. subtilis genome microarray using Affymetrix GeneChip technology (21,40). This technology offers high sensitivity, high specificity, and excellent reproducibility (19). We show that the microarray can monitor gene expression changes in response to transition from the exponential to the stationary growth phases and exposure to three different vitamins that repress expression of biosynthetic genes. Moreover, we also present evidence that the microarray can be used to detect novel transcripts within intergenic regions and on the opposite strand of known genes, leading in some cases to the identification of previous unreported coding regions. MATERIALS AND METHODSMicroarray design. An "antisense" oligonucleotide array complementary to the Baci...
In prokaryotes, in the absence of protein serine/threonine/tyrosine kinases, protein histidine kinases play a major role in signal transduction involved in cellular adaptation to various environmental changes and stresses. Histidine kinases phosphorylate their cognate response regulators at a specific aspartic acid residue with ATP in response to particular environmental signals. In this His-Asp phosphorelay signal transduction system, it is still unknown how the histidine kinase exerts its enzymatic function. Here we demonstrate that the cytoplasmic kinase domain of EnvZ, a transmembrane osmosensor of Escherichia coli can be further divided into two distinct functional subdomains: subdomain A [EnvZ(C)⅐(223-289); 67 residues] and subdomain B [EnvZ(C)⅐(290-450); 161 residues]. Subdomain A, with a high helical content, contains the autophosphorylation site, H-243, and forms a stable dimer having the recognition site for OmpR, the cognate response regulator of EnvZ. Subdomain B, an ␣/-protein, exists as a monomer. When mixed, the two subdomains reconstitute the kinase function to phosphorylate subdomain A at His-243 in the presence of ATP. Subsequently, the phosphorylated subdomain A is able to transfer its phosphate group to OmpR. The two-domain structure of this histidine kinase provides an insight into the structural arrangement of the enzyme and its transphosphorylation mechanism.
Escherichia coli osmosensor EnvZ is a protein histidine kinase that plays a central role in osmoregulation, a cellular adaptation process involving the His-Asp phosphorelay signal transduction system. Dimerization of the transmembrane protein is essential for its autophosphorylation and phosphorelay signal transduction functions. Here we present the NMR-derived structure of the homodimeric core domain (residues 223-289) of EnvZ that includes His 243, the site of autophosphorylation and phosphate transfer reactions. The structure comprises a four-helix bundle formed by two identical helix-turn-helix subunits, revealing the molecular assembly of two active sites within the dimeric kinase.
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